Western Blot for Low Molecular Weight Protein

Get Shareable Link

Abstract:      During western blot, some protein targets can be easily detected. However, western blot analysis always fails for other detection targets. Western blot result depends on protein molecular weight. How to conduct western blot for low molecular weight protein is specified.
Keywords:   Western Blot Protein, Western Blot Molecular Weight, Low Molecular Weight Protein

1. Western Blot Protein Loading Amount

Usually, the loading amount for each well is 30-50μg. The content of 80% protein in cells is very low. If the molecular weight is very low, 50-100μg is recommended.

2. Western Blot Gel Electrophoresis

Choose suitable gel concentration. 5% stacking gel is recommended for low molecular weight protein. Separating gel concentration is specified in the following table.

Molecular Weight(kDa) Separating Gel Concentration
X≤10 15%
10<X≤15 13.5%
15<X≤25 12%

Low molecular weight protein running gels at higher voltage will become serrated. It's suggested to run the stacking gel at 80V for 30 min. Later, run at 100V till the bromophenol blue (BPB) band was 1cm from the bottom of the gel.

3. Trarsmembrane

3.1. Trarsmembrane Selection

Common solid phase materials in western blot are NC membrane, DBM, DDT, nylon membrane and PVDF membrane. PVDF membrane (polyvinylidene fluoride) is recommended. Because proteins bind with PVDF membrane through hydrophobic interaction, it's easier for antibodies to bind with hydrophilic site exposed to the liquid phase.

Two PVDF membrane for proteins with different molecular weight: 0.45μm for protein(MW＞20 kDa), 0.2μm for protein(MW＜20 kDa). 0.2μm can prevent proteins from directly passing through the membrane during transfer. Soak the PVDF membrane in methanol for 5min to activate the positive group and facilitate binding with negatively charged proteins. Then, put the PVDF membrane in transfer buffer.

3.2. Transmembrane Method

Conditions of wet membrane transfer are specified in the following table.

Molecular Weight(kDa) Transmembrane Conditions
X≤10 200mA constant current for 30mins
10<X≤15 200mA constant current for 40mins
15<X≤20 200mA constant current for 50mins

The equilibration time before the transfer should be within several minutes(or even 1 min). SDS is not required for equilibrium liquid and components of transfer buffer.

4. Western Blot Ponceau Staining for Determining Protein Abundance

The stained PVDF membrane can detect western blot band size during electrophoresis. The accuracy of loading amount can be primarily evaluated by comparing the sample band in each lane.

5. Primary Antibody Dilution Ratio

Dilution ratio for low molecular weight proteins is recommended in the manual. Low dilution ratio helps antibody binding.

6. Western Blot Exposure Time

Adjust the exposure time appropriately according to signal intensity. If the band is visible in the dark place, the exposure should be within 30s-2min. If the signal is weak, the band is not clear after adding the developer. Take out of the membrane and react for a while. Then put it into the machine and add the developer again. Alternatively, a clear band can be got by extending the developing time.

Recommended Antibodies

REFERENCES

[1] Optimization of western blotting for the detection of proteins of different molecular weight, PMID: 32283940.
[2] Western blotting of high and low molecular weight proteins using heat, PMID: 26044007.